Antigen discovery has focused on using human serum samples collected from individuals that are naturally exposed to malaria and appear to develop effective immunity that prevents gametocytemia or blocks parasite transmission to mosquitoes, as tools to identify candidate vaccine antigens. Specifically, serum samples or antibodies that have activity of interest are compared to sera/antibodies that lack this activity, for their ability to select or recognize individual recombinant proteins constructs of P. falciparum. Recombinant proteins identified through differential screening are then prepared as immunogens and tested for their ability to induce effective anti-gametocyte or transmission-blocking antibodies. In FY2019, a third human monoclonal antibody was generated from the sorted B cells from subjects immunized with Pfs230 in Alhydrogel in our Mali clinical trials. This monoclonal antibody reacted to Pfs230D1 by ELISA but did not block parasites in the standard membrane feeding assay. Competition ELISA experiments determined that the 2 human antibodies against Pfs230D1 have different epitopes and bind to Pfs230D1 with similar nanomolar affinities. ELISA results showed the 2nd antibody binds to Pvs230D1 and can partially compete with the 3rd antibody. The 3rd antibody recognized Pvs230D1, suggesting that the 2nd and 3rd antibodies have different but overlapping epitopes. In FY2019, 6 new fragments of Pfs230 were constructed for expression in mammalian systems. Expression of 2 of these fragments D1-3 and D1-4 were confirmed by Western Blot reactivity to 3 human mAb against Pfs230D1. Production was scaled up and transient transfection produced milligram quantities of both fragments. Conditions for generating full-length Pfs230 in mammalian cells are being screened. In FY2019, 2 new potential TBV candidate were expressed. First glideosome associate protein (GAP50) was expressed and produced in mammalian cells. The parasite protein GAP50 recruits and binds factor H then disables the complement pathway. Antibodies to GAP50 could block factor H recruitment and enhance complement activity; therefore, pairing GAP50 with Pfs230D1 could enhance its activity. The extracellular domain of GAP50 was expressed in mammalian cells and milligram quantities were generated from a transient transfection. Second Pfs48/45D3 was expressed in drosophila cells. The protein generated in the small-scale expression experiment reacted to conformational specific antibody 3E12. Additionally, 3E12 reduced oocyst as shown by standard membrane feeding assay (SMFA), therefore our recombinant Pfs48/45D3 recapitulates an epitope that has functional activity.